1. Field of the Invention
The present invention relates to a method for designing a primer in a real-time PCR (to be hereafter referred to as RT-PCR).
2. Background Art
As one of the means for comparing the amount of expression of genes, a method whereby the amount of mRNA accumulated in cells is determined is widely employed. RT-PCR is widely utilized in recent years as a method for measuring the amount of accumulated mRNA. In a general procedure involving RT-PCR, the accumulated amount of mRNA is measured as follows. Samples grown under different conditions, or samples of different series grown under identical conditions are prepared, and mRNA is extracted from a tissue of concern. Using the thus extracted mRNA as a template, PCR is conducted under conditions including a primer set specific to a target gene and a fluorescent dye that binds to double strands to emit color. The fluorescent intensity in a reaction solution is measured in parallel with the reaction, whereby the number of nucleic acids in the double strands is measured. Based on the increase in fluorescent intensity, the amount of amplification product is determined.
PCR is a highly sensitive process and is theoretically capable of performing amplification even from a few copies of template. If genomic DNA is mixed in the extracted mRNA, amplification even occurs based on the genomic DNA as a template. In this case, PCR products would include both those based on mRNA as a template and those based on genomic DNA as a template. As a result, the amount of mRNA would not be accurately measured.
With reference to FIG. 1, the relationship between the types of template and the length of PCR products is described. In a eukaryotic experimental system, when splicing a target gene, individual primers are designed on different exons. Genomic DNA 101 has intron 102 and exon 103. Primer 104 on the sense strand side is represented by an arrow pointing to the right, and primer on the nonsense strand side by an arrow pointing to the left. When genomic DNA 101 is used as a template, a PCR product 106 would include an intron portion. When mRNA 110 is used as a template, a PCR product 111 would not include an intron portion and would therefore be shorter than when genomic DNA is used as a template. Based on the lengths of the PCR products, amplification products based on genomic DNA as a template can be distinguished from amplification products based on mRNA as a template. For this purpose, a method is required for designing a primer on exons with an intron portion disposed between them.
Furthermore, when the measured mRNA amount is compared between different samples, an analytical curve must be prepared. Theoretically, if the temperature cycle in PCR differs by one cycle, the amount of template doubles. However, in practice, this varies from one experiment to another, depending on experimental conditions, target base sequence, and primer sequence. Thus, for comparing the mRNA amount between different samples, a PCR product is prepared that includes only the target base sequence of the primer, and then an analytical curve is prepared using the dilution series of DNA that contains only the target base sequence as a template. Accordingly, a method is required for designing a primer for preparing a template that only contains the target base sequence of the primer for measuring the mRNA amount.    Patent Document 1: JP Patent Publication (Kokai) No. 2001-245697 A